WO2000018950A2 - Procede d'analyse haut rendement pour enzymes hydrolysant metaboliquement des triphosphates nucleosidiques et systeme d'analyse utilisant ce procede - Google Patents

Procede d'analyse haut rendement pour enzymes hydrolysant metaboliquement des triphosphates nucleosidiques et systeme d'analyse utilisant ce procede Download PDF

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WO2000018950A2
WO2000018950A2 PCT/US1999/021365 US9921365W WO0018950A2 WO 2000018950 A2 WO2000018950 A2 WO 2000018950A2 US 9921365 W US9921365 W US 9921365W WO 0018950 A2 WO0018950 A2 WO 0018950A2
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enzyme
assay
adp
target enzyme
ndp
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WO2000018950A3 (fr
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Anthony R. Welch
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Bioqual Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is directed to a method of assaying enzymes which metabolically hydrolyze nucleoside triphosphates and further to an assay system for carrying out the disclosed method.
  • NTPs nucleoside triphosphates
  • All known organisms contain enzymes which hydrolyze nucleoside triphosphates (NTPs) as cosubstrates of biochemical reactions.
  • NTPs nucleoside triphosphates
  • the hydrolysis of the phosphoanhydride bonds of NTPs is very exergonic, i.e. the hydrolysis of the bonds releases free energy and the reactions are thus thermodynamically favored.
  • the energy released upon hydrolysis of NTPs is used by an organism to drive many endergonic reactions, reactions which are thermodynamically unfavored.
  • NTP hydrolyzing enzymes are attractive candidates for drug targeting.
  • one of the limiting steps in drug design is the necessity for a high throughput assay which can be used to quickly screen potential inhibitors (or activators) of specific enzymes.
  • Enzyme screening assays are typically based on a method of quantitating the metabolic formation of enzyme products.
  • the specific nature of the products generated by each specific enzyme reaction that hydrolyzes an NTP as a cosubstrate dictates that individualized assay methods for quantitating the specific enzyme product produced have to be developed for each different enzyme of interest.
  • Individualized assay design is both time-consuming and expensive.
  • the present invention provides a high throughput robust assay method for studying enzymes, which hydrolyze NTPs.
  • the present assay method further provides a universal reporter system such that diverse enzymes which hydrolyze NTPs can be screened with the same assay format. This will allow for easy roboticization of the assay and high throughput screening for compounds, particularly inhibitors, of interest.
  • the present invention is drawn to a method of screening for inhibitors or activators of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises,
  • NDP NDP, where NDP is a nucleoside diphosphate
  • the present invention further encompasses a method of screening for inhibitors or activators of enzymes which hydrolyze a nucleoside triphosphate as a cosubstrate which comprises ,
  • FIG. 1 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents only) .
  • FIG. 1 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and PPi cosubstrate) .
  • Figure 3 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and ADP-D-glycero-D-mannoheptose target substrate) .
  • FIG. 4 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents and ADP-heptose synthetase target enzyme) .
  • FIG. 1 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi and ADP-D-glycero-D-mannoheptose) .
  • FIG. 1 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi and ADP-heptose synthetase) .
  • FIG. 7 ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, ADP-D-glycero-D-mannoheptose and ADP- heptose synthetase) .
  • Figure 8. ADP-heptose synthetase reaction coupled to luciferase/luciferin reporter enzyme (luciferase/luciferin reporter reagents, PPi, ADP-D-glycero-D-mannoheptose and ADP- heptose synthetase) .
  • ADP-heptose synthetase demonstrates Michaelis- Menton kinetics with respect to the target substrate, ADP-D- glycero-D-mannoheptose .
  • Figure 10 Linear relationship between v . and the concentration of the target enzyme, ADP-heptose synthetase.
  • Figures 13A and B show that the velocity of the luciferase reaction (defined as RLU/min from 5-40 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, CDP-choline.
  • Figure 13B shows that a Lineweaver-Burk plot of 1/ [S] vs. l vi demonstrates a linear correlation.
  • the direction of an enzymatic reaction is defined based on the metabolic pathway in which a specific enzyme participates in a cell.
  • the metabolic forward direction is the ultimate target for inhibition or activation by compounds identified with the present invention.
  • all biochemical reactions are reversible with the reverse reaction occurring at the same reaction site as the forward reaction.
  • the metabolic products of the forward direction become the substrates for the reverse reaction.
  • enzymes which hydrolyze NTPs as cosubstrates in the metabolic forward direction will synthesize the respective NTP as a product if the reaction is thermodynamically pushed in the reverse reaction.
  • the present invention is drawn to a high throughput assay method of monitoring the activity of enzymes which hydrolyze NTPs as cosubstrates by forcing the reaction to run in the metabolically reverse direction and quantitating the NTP synthesized by the target enzyme.
  • the NTPs synthesized by the reverse reaction can be universally quantitated by monitoring the activity of a coupled enzyme, known as a reporter enzyme, which uses ATP as a cosubstrate to produce light, color or some other measurable readout .
  • An important aspect of the present assay method is that the method of ATP quantification be enzyme-dependent .
  • the enzyme reaction catalyzed by the reporter enzyme will be thermodynamically favored such that the reaction catalyzed by the target enzyme will be pulled in the metabolic reverse direction to synthesize, rather than hydrolyze NTPs.
  • the present invention provides a single format high throughput robust assay method for screening for inhibitors or activators with all enzymes which metabolically hydrolyze NTPs.
  • the reporter enzyme system of the present invention is an enzyme read out system, which uses ATP as a cosubstrate in the enzymatic generation of a signal. From a kinetic perspective, the reporter enzyme reaction is only rate limited by the production of ATP by the target enzyme system. As a result the reporter enzyme thermodynamically pulls the target enzyme in the reverse direction while at the same time providing a read out system for quantitating target enzyme activity.
  • the general design of an assay of the present invention will involve first identifying a target enzyme.
  • the target enzyme may be any enzyme of interest which hydrolyzes an NTP in the metabolic forward direction.
  • the product or products, which are produced by the target enzyme reaction in the metabolic forward direction also need to be identified.
  • the product (s) produced by the metabolic forward reaction, or analogues thereof, will be used as substrates and cosubstrates in reverse reaction.
  • optimum reaction conditions can be determined as detailed below.
  • the present invention can be adapted to create a universal high throughput method for use with all target enzymes which hydrolyze NTPs, regardless of the NTP cosubstrate, by coupling the reverse reaction of the target enzyme to a nucleoside diphosphate kinase driven reaction.
  • the nucleoside diphosphate kinase catalyzes the transfer of a phosphate group from the NTP produced by the reverse target enzyme reaction to ADP, producing ATP, which can be quantitated by the reporter enzyme as described above.
  • Assay Scheme (I) Assay Scheme (I) as follows: Assay Scheme (I) :
  • the reverse target enzyme reaction is itself first coupled to nucleoside diphosphate kinase.
  • the nucleoside diphosphate kinase will transfer a phosphate group from the NTP synthesized in the target enzyme reverse reaction, to ADP, generating ATP.
  • the ATP can then be quantitated by the reporter enzyme system.
  • Assay Scheme (II) The reaction scheme for the assay method of the present invention with target enzymes which produce NTPs other than ATP is depicted as Assay Scheme (II) , as follows:
  • Enzymes which metabolically hydrolyze NTPs can be classified into four general groups based on which bond within the NTP is cleaved.
  • Group I enzymes are protein, lipid, carbohydrate, amino acid, or other organic molecule kinases which catalyze the following general reaction (I) :
  • X is a protein, lipid, carbohydrate, amino acid or other organic molecule having a nucleophilic hydroxyl group.
  • Nuc represents the nucleophilic functionality of X. Typically, the nucleophilic functionality of X is a hydroxyl moiety. Lipids, proteins, carbohydrates and amino acids may be naturally occurring or synthetic and have their standard chemical definition as found, for example, in Stenesh, J. , Dictionary of Biochemistry and Molecular Biology, John Wiley & Sons, Inc. (1989) . This reaction involves the nucleophilic
  • the Group I enzymes use X-Nuc-P as the target substrate in the reverse reaction, with the appropriate NDP as a cosubstrate.
  • Group I enzymes which use ATP as a cosubstrate and thus may be used in Assay Scheme (I) , above, include protein kinases as generally disclosed in Hunter T, eth. in Enzymology, 200: 3-37 (1991), such as protein kinase C and protein kinase A; lipid kinases such as ethanolamine kinase, Weinhold and Rethy, Biochem. 13: 5135-5141 (1974) and choline kinase, McCaman et al . , Anal. Biochem. 42: 171-177; ca'rbohydrate kinases such as hexokinase, Schwartz and Basford, Biochem.
  • protein kinases as generally disclosed in Hunter T, eth. in Enzymology, 200: 3-37 (1991), such as protein kinase C and protein kinase A; lipid kinases such as ethanolamine kinase, Weinhold and Rethy, Bio
  • kinases such as phosphoglycerate kinase, Scopes Biochem. J. 113:551-554(1969); glycerol kinase, Hayashi and Lin, J. Biol. Chem. 242:1030-1035(1967); mealonate-5-phosphotransferase and phosphomevalonate kinase, Bazas and Beytia, Biochem. 19:2300- 2304 (1980) ; and flavokinase, Mayhew and Wassink, Biochim. Biophys. Acta 482:341-347 (1977).
  • Assay Scheme II may be used to measure enzyme activity and screen for inhibitors or activators.
  • the NTP produced by the reverse target enzyme reaction is utilized as a substrate in a reaction with ADP and the enzyme nucleoside diphosphate kinase.
  • the nucloeside diphosphate kinase transfers the phosphate group from the NTP to the ADP, producing ATP which, in turn, can be monitored by the reporter enzyme.
  • the nucleoside diphosphate kinase When using Assay Scheme II, the nucleoside diphosphate kinase is saturated with ADP such that the amount of ATP produced by the nucleoside diphosphate kinase from ADP is directly proportional to the amount of NTP produced by the reverse target enzyme reaction.
  • Group I target enzymes which may be used with Assay Scheme II include: when NTP is GTP; phosphoenolpyruvate carboxykinase (PEPCK) , Ballard and Hanson, J. Biol. Chem. 244:5625- 5630(1969) and nucleoside diphosphate kinase, Edlund, Acta Chem. Scand. 25:1370-1376 (1971);
  • Group II enzymes are nucleotide-sugar synthetases or nucleotide lipid synthetases, nucleotide transferases, or pyrophosphorylases which metabolically catalyze the following reaction (II) :
  • reaction (II) X-Nuc is typically a phosphorylated carbohydrate or lipid molecule.
  • Reaction (II) involves a
  • the substrate of the reverse reaction of Group (II) enzymes is NDP-X, with PPi as a cosubstrate.
  • Examples of Group (II) target enzymes include:
  • nucleotide sugar sythetases such as ADP-heptose synthetase, which is encoded by the rfaE gene of gram negative bacteria, Ding et al . J. Biol. Chem. 269: 24384- 24390 (1994)
  • FAD pyrophosphorylase which synthesizes FAD from riboflavin, Gomes and McCormick, Proc . Soc . Exp. Med. 172 :250-254 (1983) ;
  • bacterial Group (II) enzymes for example, rfaE enzyme, as discussed above, of Helicobacter pylori , which provide ideal targets for drug therapies.
  • Group (III) enzymes are cyclases which catalyze the following reaction (III) : cyclase NTP « -» CNMP + PP j
  • the substrate for the reverse reaction is cyclic NMP (cNMP) , with PPi as a cosubstrate.
  • Groups III enzymes include adenylate cyclase, Tao and Lipman, Proc. Natl. Acad. Sci. USA 63:86-92 (1969), which uses cAMP in the reverse reaction as a substrate; guanylate cyclase, White and Zenser, Meth. in Enzymol . 38:192-195 (1979), which uses cGMP as a substrate in the reverse reaction.
  • Group IV enzymes transfer the adenosyl group of ATP in the following general reaction (IV) :
  • Group IV enzymes are responsible for the synthesis of coenzyme B 12 (where X is Co + ) and S-adenosylmethionine (where X is methionine) .
  • Group IV enzymes include, cobalamin synthetase,
  • the reporter enzyme system is based on an enzyme which uses ATP as a cosubstrate and which produces some measurable signal upon hydrolysis of the ATP.
  • reporter enzymes include a luciferase/luciferin based system or a phosphoglycerate kinase and glyceraldehyde 3 -phosphate dehydrogenase system, as detailed in Hasen et al . Meth. in Enzymol . 8:248 (1966).
  • the latter system utilizes the conversion of NADH to NAD+ by the dehydrogenase and NAD+ production may be monitored spectrophormetrically .
  • Firefly luciferase catalyzes the following reaction.
  • one of the products produced upon the hydrolysis of ATP is light.
  • the amount of light produced can be easily quantitated using a luminometer and is directly related to the amount of ATP hydrolyzed by the luciferase when the luciferin substrate is present at saturating amounts. Since the Km of luciferin for luciferase
  • luciferase will be greater than 90% saturated at the minimum concentration of 63-
  • luciferase/luciferin is the reporter enzyme
  • General reaction conditions for the assay in which luciferase/luciferin is the reporter enzyme include a buffered solution between pH 6.5 - 8.5, with peak activity at pH 7.8 (Lundin et al . , Anal. Biochem. 75:611-620 (1976); 5-10 mM Mg +2 ;
  • the reaction may also optionally
  • the buffer conditions will be adjusted as appropriate for each target enzyme tested. It will be readily apparent to one skilled in the art how to modify the buffer conditions, with regard to buffer composition, pH and serum albumin as necessary to accommodate the reaction conditions of the target enzyme. Ideally, care should be taken to keep the luciferase reaction as near pH 7.8 as possible since at this pH it will be more active and will therefore thermodynamically pull the reaction of the target enzyme in reverse. Changes in luciferin and luciferase concentrations can alter the sensitivity of the assay. To validate that the concentrations of luciferin and luciferase used are behaving properly as a reporter system, each assay should be evaluated to ensure that the velocity of the luciferase reaction (i.e. light produced per unit time) is directly proportional to the target enzyme concentration . Exemplified reaction conditions are as follows: Gylcine - 100 mM
  • ImM EDTA may optionally be included in the reaction.
  • the reaction mixture will ideally be substantially free from exogenous NTPs. "Substantially free from exogenous NTPs" means that the components of the reaction mixture, i.e. the target enzyme, target cosubstrate, target substrate, reporter enzyme system etc., does not need to be completely pure, however, no more than negligible amounts (i.e. no more than 1
  • NTPs should be present. As such, the reaction should be evaluated for exogenous contamination of NTPs. This may be easily done by separately introducing each target substrate or cosubstrate into the reporter enzyme reaction mixture in the absence of the target enzyme, to assess for contamination of the target substrate or cosubstrate. To assess for possible contamination of the target enzyme, the target enzyme should be added to the reaction mixture in the absence of substrate and cosubstrate. In addition, the target enzyme must be saturated with the appropriate cosubstrates such that the reverse reaction rate is linear with respect to the target substrate concentration.
  • a general reaction mixture for the present inventive method using luciferase as reporter enzyme with a target enzyme that metabolically hydrolyzes ATP includes in an appropriate buffer: a) target enzyme or target substrate; b) saturating concentrations of appropriate cosubstrates;
  • the reaction can be initiated with the addition of target substrate or target enzyme and the rate of ATP synthesis, which is directly correlated to the reaction catalyzed at the active site of the target enzyme, measured with a luminometer as light generated per unit time.
  • the assay should be run under conditions where the concentration of the target substrate is less than the K M of the target substrate for the target enzyme under given reaction conditions.
  • the K M of the target substrate for the target enzyme can be determined experimentally by evaluating the amount of light produced per unit time (initial velocity) as a function of target substrate concentration. See Fersht , Enzyme Structure and Mech . , 2 nd Ed, 1985, W.H. Freeman and Co. NY.
  • the initial velocity of the reaction will be proportional to the concentration of target substrate.
  • reaction rate is linear and l ⁇ M is a valid
  • the linear response of the luciferase reporter enzyme should be demonstrated to be directly proportional to the amount of target substrate and target enzyme used in the screening assay. This can be done, by simply doubling or halving the target substrate or target enzyme concentration and evaluating the initial velocity.
  • the reporter system When the target enzymes utilizes an NTP other than ATP as a substrate, the reporter system will be coupled to nucleoside diphosphate kinase to produce ATP from the target enzyme NTP product.
  • the nucleoside diphosphate kinase coupled reporter system is used, ADP and nucleoside diphosphate kinase are additionally added to the reaction system.
  • concentration will typically be 20-200 ⁇ M with 1-100 U/ml
  • nucleoside diphosphate kinase in particular, the ADP
  • concentrations of ADP may inhibit the luciferase reaction, Denberg et al . , Arch. Biochem. Biophys. 134:381-394(1969).
  • the reverse reaction catalyzed by Group I enzymes can be further encouraged thermodynamically by the addition of the enzyme inorganic pyrophosphotase to either Assay Scheme I or II when the reporter enzyme is luciferase.
  • Inorganic pyrophosphotase catalyzes the hydrolysis of PPi into 2Pi. Since this involves the hydrolysis of a phosphoanhydride bond in PPi there is significant free energy released upon cleavage.
  • PPi is a product of the luciferase reaction, hence coupling the hydrolysis of this product to Assay Scheme I or II will contribute additional thermodynamic "pull" at driving the reverse reaction of the target enzyme.
  • the present examples embody assays for screening for inhibitors or activators of Group II enzymes.
  • the following examples are directed to evaluating the enzymatic activity of an ADP-heptose synthetase, a CTP :phosphocholine cytidylyltransferase, and an N-acetylgluocosamine-1-phosphate uridylyltransferase
  • the present formats may be used for any Group II enzymes.
  • Bacteria typically contain unique enzymes which hydrolyze nucleoside triphosphates as cosubstrates and which are involved in biosynthetic pathways which are critical to bacterial survival. As such, these unique enzymes provide an ideal target for drug therapy.
  • biosynthetic pathways which produce the lipopolysaccharide (LPS) outer membrane of gram negative bacteria.
  • LPS lipopolysaccharide
  • the LPS membrane of gram negative bacteria is made from lipopolysaccharide, lipid and protein. This outer coat makes gram negative bacteria particularly refractory to standard antibiotics.
  • LPS has been shown to be directly related to the ability of the outer membrane to serve as a functional barrier to antibiotics, bile salts and other hydrophobic molecules.
  • An essential component of the LPS is L-glycero-D-mannoheptose.
  • L-glycero-D- mannoheptose is unique to gram negative bacteria.
  • the unique structure of the L-glycero-D-mannoheptose suggests that the enzymes which synthesize the molecule are likely also unique, making them ideal drug targets. Mutations in the biosynthetic enzymes responsible for inner core polysaccharides of the gram negative bacteria E. coli , S . typhi urium and H.
  • influenza demonstrate hypersensitivity to numerous antibiotics, bile salts, serum killing and temperature. These finding support that inhibition of the biosynthetic enzymes involved with LPS formation increases the sensitivity to hydrophobic antibiotics and macrolides such as vancomycin for use against gram negative bacteria.
  • ADP-heptose synthetase metabolically catalyzes the transfer of AMP to D-glycero-D-mannoheptose-lP generating ADP- D-glycero-D-mannoheptose as shown in the following reaction.
  • H. pylori is a gram negative, spiral bacteria that is a common bacteria of the human gastrointestinal tract. Infection with H. pylori is associated with most duodenal and gastric ulcers and is a risk factor in the development of gastric adenocarcinoma.
  • the present invention is therefor useful for screening potential inhibitors of ADP-heptose synthetase of H. pylori .
  • the reverse enzyme reaction of rfaE was developed as follows.
  • Recombinant ADP-heptose synthetase encoded by the rfaE gene of H. pylori was expressed in E. coli as a fusion protein with glutathione S-transferase (GST, Pharmacia, Piscataway, NJ) .
  • GST glutathione S-transferase
  • the fusion protein was purified by chromatography on a glutathione column and the ADP-heptose synthetase enzyme was removed from the GST fusion partner by specific proteolytic cleavage between the two proteins using the enzyme PreScission Protease (Pharmacia, Piscataway, NJ) .
  • ADP-heptose synthetase was estimated by quantitating the protein concentration of the purified enzyme and calculating molarity using a molecular weight of 52,656 g/mole for the enzyme.
  • ATP synthesis was evaluated by the reporter enzyme system consisting of luciferase/luciferin.
  • the cosubstrates of the reverse reaction are NDP-X (in the case of ADP-heptose synthetase, NDP-X is ADP-D-glycero-D-mannoheptose) and PPi.
  • concentrations of each of the assay components were as
  • ATP is synthesized by ADP- heptose synthetase under conditions where the reaction is run m reverse with both cosubstrates of the reaction and the enzyme present (Fig. 8) .
  • Figures 1-7 demonstrate the lack of ATP synthesis as reported by the luciferase/luciferin system when any of the components of the reaction are not added to the reaction mixture.
  • figures 1-7 demonstrate the absence of contaminating ATP m each component of the coupled assay reaction mix.
  • Each figure shown reports ATP synthesis as directly related to the amount of light formed per unit time.
  • RLU represents relative light units. The components in each reaction mixture are indicated above each relevant figure.
  • reaction conditions were similar to those reported in Experiment 1 with the exception that the concentration of ADP- D-glycero-D-mannoheptose was evaluated at the following
  • concentration of ADP-D-glycero-D-mannoheptose was determined in duplicate and the mean rate was graphed vs. concentration (Fig. 9) .
  • FIG. 9A shows that the velocity of the luciferase reaction (v defined as RLU/min over the initial 4 min of the reaction) demonstrates saturation kinetics with respect to the concentration of the target substrate, ADP-D-glycero-D- mannoheptose.
  • the combination of the data in Fig. 9A and 9B is consistent with the target enzyme demonstrating typical Michaelis-Menton kinetics with respect to the target substrate at saturating levels of PPi.
  • the K M of the target substrate for the target enzyme is 6.2 ⁇ M as determined by the
  • Table II Mean initial velocity ⁇ v ) at varying concentrations of ADP-D-glycero-D-mannoheptose.
  • the present examples embody assays for screening inhibitors or activators of enzymes that hydrolyze an NTP other than ATP.
  • Assay Scheme (II) the reverse reaction of a target enzyme which qualifies for this scheme will result m the synthesis of a specific NTP other than ATP as a product of the reverse reaction.
  • NDK nucleoside diphosphate kinase
  • CTP phosphocholme cytidyltransferase
  • UTP N-acetylglucosoamme-1- phosphate uridylyltransferase
  • CTP phosphochol ⁇ ne cytidyltransferase
  • the present format may be used for any enzyme hydrolyzmg a CTP as the cosubstrate or, more generally, any enzyme hydrolyzmg an NTP other than ATP as a cosubtrate .
  • CTP rphosphocholme cytidyltransferase is a critical enzyme m the biosynthesis of phosphatidylcholme, an essential component of many prokaryotic and eukaryotic membranes. Inhibition of the activity of this enzyme has been demonstrated to be responsible for the anti-neoplastic effect of the drugs edelfosme (Vogler, et al . 1996, Leukemia Research, 20: 947-951) and hexadecylphosphocholme (Boggs, et al . 1998, Biochim Biophys Acta, 1389: 1-12).
  • This enzyme catalyzes the transfer of a CMP group from CTP to phosphocholme to generate CDP-cholme, with the subsequent loss of pyrophosphate (PP as shown below (We hold et al . 1986, 261: 5104-5110):
  • the enzyme CCT has been shown to be composed of a number of functional domains (Kent 1997, Biochim Biophys Acta 1348: 79- 90) .
  • the full-length CCT enzyme requires lipids for in vi tro activity.
  • the necessity for lipid activation has been explained by the observation that the C-termmal domain of CCT behaves as an inhibitor of enzyme activity in the absence of lipids (Wang and Kent, 1995, J. Biol. Chem. 270: 18948-18952).
  • CT236 C-termmal truncated form of CCT
  • the truncated CT236 form of the CCT enzyme was used m the present studies.
  • an assay of Scheme II format the following enzyme coupled reaction was designed:
  • CTP CTP +0 2 + PP i + CO 2 + light
  • a product of the reverse reaction of CT236 is CTP.
  • CTP is converted to ATP by the action of the enzyme nucleoside diphosphate kinase.
  • ATP synthesis is reported using the enzyme luciferase, which produces light relative to the amount of ATP.
  • the amount of light generated is quantitated using a luminometer.
  • CT236, is important that the activity of the enzyme of interest, in this case CT236, is in fact the rate- limiting step in the coupled reactions. To insure that this is the case, the substrates of both NDK (i.e. ADP) and luciferase (i.e. luciferin) must be at or near saturating conditions .
  • luciferase 98 mM glycine, 163 mM Tris, 9.75 mM MgS0 4 , 0.975 EDTA, 2 mg/ml human serum albumin, 2 units diphosphate
  • nucleoside kinase 100 ⁇ M ADP, 1 mM sodium pyrophosphate.
  • the pH was approximately 7.8.
  • concentrations of the target substrate, CDP-choline, and the target enzyme, CT236 were varied in each experiment as specified.
  • Figure 11 demonstrates that the reaction catalyzed by the enzyme CT236 can be run in reverse under the conditions described and the rate of that reaction can be monitored using a coupled enzyme assay as described above.
  • the reaction was evaluated with the core components described above and 1 mM CDP-choline as the target substrate in the absence (solid
  • Experiment 2 defined the concentration range in which the reaction catalyzed by the target enzyme is linear with the production of light by the luciferase reporter enzyme system.
  • concentration of target substrate be below K M , in a range such that activity is linear with respect to substrate concentration. This is important in the identification of competitive inhibitors of the target substrate at low concentrations of inhibitor.
  • RLU/sec velocity of the luciferase reaction
  • FIG. 13A shows that the velocity of the luciferase reaction (defined as RLU/mm from 5-40 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, CDP- choline.
  • Figure 13B shows that a Lineweaver-Burk plot of 1/ [S] vs. l/v demonstrates a linear correlation.
  • the combination of data in Figures 13A and 13B is consistent with the target enzyme demonstrating typical Michaelis- Menton kinetics with respect to the target substrate at saturating levels of pyrophosphate (1 mM) .
  • CT236, is 120 ⁇ M. Therefore for the reverse reaction of
  • CDP-choline less than approximately 40 ⁇ M
  • the present format may be used for any enzyme hydrolyzing a UTP as the cosubstrate or, more generally, any enzyme hydrolyzing an NTP other than ATP as a cosubtrate .
  • Peptidoglycan is the primary structural polymer of the bacterial cell wall and is essential for the structural integrity of both gram negative and gram positive bacteria. Many of the most successful broad-spectrum antibiotics (e.g. penicillins, cephalosporms, vancomycm, cycloserme) inhibit peptidoglycan synthesis.
  • UDP-N-acetylglucosamme UDP-GlcNAc
  • synthesis of UDP-GlcNAc is catalyzed by the protein product of the GlmU gene.
  • the GlmU protein is a 456 ammo acid bifunctional enzyme that contains both acetyltransferase and uridylyltransferase activities (Mengm-Lecreulx and van Hei enoort, 1993, J. Bacteriol . 175- 6150-6157, Mengm-Lecreulx and van Heijenoort, 1994, J. Bacteriol . 176: 5788-5795) .
  • This enzyme catalyzes the following overall reaction:
  • Recombinant protein encoded by the GlmU gene of the gram negative pathogen, Helicobacter pylori was expressed in E. coli as a fusion protein with glutathione-S- transferase (GST, Pharmacia, Piscataway, NJ) .
  • GST glutathione-S- transferase
  • the fusion protein was purified by chromatography on a glutathione-sepharose column and the GlmU enzyme was removed from the GST fusion partner by specific proteolytic cleavage between the two proteins using the enzyme PRESCISSION Protease (Pharmacia, Piscataway, NJ) .
  • a product of the reverse reaction of the GlmU uridylyltransferase activity is UTP
  • UTP is converted to ATP by the action of the enzyme nucleoside diphosphate kinase (NDK) .
  • NDK nucleoside diphosphate kinase
  • ATP synthesis is reported using the enzyme luciferase, which produces light relative to the amount of ATP.
  • the amount of light generated is quantitated using a lummometer.
  • the target enzyme is the GlmU uridylyltransferase and the target substrate is UDP-GlcNAc. Therefore, to insure that the target reaction is the rate limiting reaction, the concentration of PP ⁇ ADP and luciferin must be at or near saturating conditions.
  • UTP synthesis was measured by coupling the production of UTP to the synthesis of ATP using the enzyme NDK as depicted above. ATP synthesis was evaluated by quantitating the amount of light produced by the luciferase/luciferin reporter enzyme system on a luminometer. The concentration of each of the
  • assay core components were as follows: 130 ⁇ M luciferin, 20
  • nucleoside kinase 100 ⁇ M ADP, 1 mM sodium pyrophosphate.
  • Figure 14 demonstrates that the GlmU uridylyltransferase reaction can be reversed to synthesize UTP using the coupled assay design shown above.
  • the activity shown is dependent on the presence of both the target substrate and target enzyme in the assay (closed triangles) . In the absence of either target substrate (open triangles) or target enzyme (closed circles) , no light is observed in the coupled assay.
  • Experiment 2 - GlmU uridylyltransferase activity is linear with respect to light production m the coupled assay at
  • concentrations of enzyme between 0.28 ⁇ M and 1.12 ⁇ M.
  • the UDP-GlcNAc concentrations evaluated were 5, 10, 20, 40, 80, 130, 160, 320, and 520
  • the initial velocity was determined by evaluating
  • Table VI shows v at each concentration evaluated.
  • Figure 16 shows that the velocity of the luciferase reaction (defined as RLU/min from 4-64 sec) demonstrates saturation kinetics with respect to the concentration of target substrate, UDP- GlcNAc.
  • SigmaPlot curve fit the above data was fit to a rectangular hyperbola:
  • the enzyme activity and can be used to screen inhibitors.
  • Protein kinases catalyze the transfer of the ⁇ -phosphoryl
  • Protein kinase A also known as cAMP-dependent protein kinase
  • Protein kinase A transfers the ⁇ -
  • Protein kinase A is an intracellular enzyme containing two domains, a regulatory and a catalytic domain. The regulatory domain is controlled by the binding of cyclic AMP (cAMP) .
  • cAMP cyclic AMP
  • a specific peptide substrate of Protein kinase A has the sequence Leu-Arg-Arg-Ala-Ser-Leu-Gly and is referred to as "kempeptide" (Mailer et al . , Proc. Natl. Acad. Sci. USA 75:248 (1978) ) . Therefore, the protein kinase A reaction using kempeptide as a substrate can be drawn:

Abstract

La présente invention concerne un procédé d'analyse haut rendement permettant de contrôler l'activité d'enzymes qui hydrolysent des triphosphates nucléosidiques (NTP) comme cosubstrats. Ce procédé consiste à forcer la réaction à se dérouler dans le sens inverse à celui du métabolisme, puis à quantifier les NTP synthétisés par l'enzyme cible. La présente invention concerne également des systèmes d'analyse utilisant le procédé susmentionné.
PCT/US1999/021365 1998-09-25 1999-09-17 Procede d'analyse haut rendement pour enzymes hydrolysant metaboliquement des triphosphates nucleosidiques et systeme d'analyse utilisant ce procede WO2000018950A2 (fr)

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Publication number Priority date Publication date Assignee Title
CN108330160A (zh) * 2018-04-18 2018-07-27 易尚明天科技有限公司 核苷二磷酸激酶ndpk活性的检测方法
CN109182564A (zh) * 2018-08-24 2019-01-11 上海芯超医学检验所有限公司 一种检测分析幽门螺旋杆菌的毒性及致病性的方法
CN111971049A (zh) * 2018-06-14 2020-11-20 北京生命科学研究所 促进免疫应答

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FR2699930A1 (fr) * 1992-12-24 1994-07-01 Kabore Paul Réactifs et procédés pour le dosage d'éléments réactifs et autres cofacteurs d'une réaction.
WO1994017198A1 (fr) * 1993-01-22 1994-08-04 Regents Of The University Of Minnesota Titrage fluorometrique enzymatique de la cyclase adenylate
EP0794260A1 (fr) * 1996-03-04 1997-09-10 Kikkoman Corporation Réactif bioluminiscent et méthode pour la détermination quantitative d'esters phosphatiques de l'adénosine en employant ce réactif
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FR2699930A1 (fr) * 1992-12-24 1994-07-01 Kabore Paul Réactifs et procédés pour le dosage d'éléments réactifs et autres cofacteurs d'une réaction.
WO1994017198A1 (fr) * 1993-01-22 1994-08-04 Regents Of The University Of Minnesota Titrage fluorometrique enzymatique de la cyclase adenylate
US5741635A (en) * 1996-01-30 1998-04-21 Mount Sinai Hospital Corporation Method of quantitating GTP and GDP bound to a G protein and uses thereof
EP0794260A1 (fr) * 1996-03-04 1997-09-10 Kikkoman Corporation Réactif bioluminiscent et méthode pour la détermination quantitative d'esters phosphatiques de l'adénosine en employant ce réactif

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108330160A (zh) * 2018-04-18 2018-07-27 易尚明天科技有限公司 核苷二磷酸激酶ndpk活性的检测方法
CN111971049A (zh) * 2018-06-14 2020-11-20 北京生命科学研究所 促进免疫应答
CN109182564A (zh) * 2018-08-24 2019-01-11 上海芯超医学检验所有限公司 一种检测分析幽门螺旋杆菌的毒性及致病性的方法
CN109182564B (zh) * 2018-08-24 2021-05-25 上海芯超医学检验所有限公司 一种检测分析幽门螺旋杆菌的毒性及致病性的方法

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